Use of VEGF and homologues to treat neuron disorders

ABSTRACT

The present invention relates to neurological and physiological dysfunction associated with neuron disorders. In particular, the invention relates to the involvement of vascular endothelial growth factor (VEGF) and homologues in the aetiology of motor neuron disorders. The invention further concerns a novel, mutant transgenic mouse (VEGF m/m ) with a homozygous deletion in the hypoxia responsive element (HRE) of the VEGF promoter which alters the hypoxic upregulation of VEGF. These mice suffer severe adult onset muscle weakness due to progressive spinal motor neuron degeneration which is reminiscent of amyotrophic lateral sclerosis (ALS)—a fatal disorder with unknown aetiology. Furthermore, the neuropathy of these mice is not caused by vascular defects, but is due to defective VEGF-mediated survival signals to motor neurons. The present invention relates in particular to the isoform VEGF 165  which stimulates survival of motor neurons via binding to neuropilin-1, a receptor known to bind semaphorin-3A which is implicated in axon retraction and neuronal death, and the VEGF Receptor-2. The present invention thus relates to the usage of VEGF, in particular VEGF 165 , for the treatment of neuron disorders and relates, in addition, to the usage of polymorphisms in the VEGF promotor for diagnosing the latter disorders.

FIELD OF THE INVENTION

The present invention relates to neurological and physiologicaldysfunction associated with neuron disorders. In particular, theinvention relates to the involvement of vascular endothelial growthfactor (VEGF) and homologues in the aetiology of motor neuron disorders.The invention further concerns a novel, mutant transgenic mouse(VEGF^(m/m)) with a homozygous deletion in the hypoxia responsiveelement (HRE) of the VEGF promoter which alters the hypoxic upregulationof VEGF. These mice suffer severe adult onset muscle weakness due toprogressive spinal motor neuron degeneration which is reminiscent ofamyotrophic lateral sclerosis (ALS)—a fatal disorder with unknownaetiology. Furthermore, the neuropathy of these mice is not caused byvascular defects, but is due to defective VEGF-mediated survival signalsto motor neurons. The present invention relates in particular to theisoform VEGF₁₆₅ which stimulates survival of motor neurons via bindingto neuropilin-1, a receptor known to bind semaphorin-3A which isimplicated in axon retraction and neuronal death, and the VEGFReceptor-2. The present invention thus relates to the usage of VEGF, inparticular VEGF₁₆₅, for the treatment of neuron disorders and relates,in addition, to the usage of polymorphisms in the VEGF promotor fordiagnosing the latter disorders.

BACKGROUND OF THE INVENTION

VEGF is a key player in the formation of new blood vessels(angiogenesis) during embryonic development as well as in a variety ofpathological conditions^(1,2). Although VEGF primarily stimulatesendothelial cells, it may also act on other cell types. Indeed, VEGF,VEGF receptor-1 (VEGFR-1/Flt1) and VEGF receptor-2 (VEGFR-2/KDR/Flk1)have recently been implicated in stroke^(3,4), spinal cord ischemia⁵,and in ischemic and diabetic neuropathy⁶, WO 0062798. However, thelatter molecules act predominantly via affecting vascular growth orfunction and a direct effect of VEGF on for example neuronal cells hasnot been shown^(11, 12). Moreover, the in vivo relevance of such adirect effect is not validated.

Ischemia plays an essential role in the pathogenesis of neurologicaldisorders, acutely during stroke and chronically during aging andseveral neurodegenerative disorders such as Alzheimer's disease,Parkinson's disease and Huntington disease. Neurons are particularlyvulnerable to oxidative stress by free radicals (generated duringischemia/reperfusion) because of their high oxygen consumption rate,abundant lipid content, and relative paucity of antioxidant enzymescompared to other organs¹⁶. Cumulative oxidative damage due to a toxicgain of function of mutant Cu, Zn-superoxide dismutase (SOD1)participates in degeneration of motor neurons in a number of patientswith familial amyotrophic lateral sclerosis (ALS)^(17,18). ALS affects 5to 10 individuals per 100,000 people worldwide during the second half oftheir life, is progressive, usually fatal within 5 years after onset ofsymptoms, and untreatable¹⁷⁻¹⁹. Ninety to 95% of cases are sporadic.Although the mechanisms underlying sporadic ALS remain unknown, evidencesuggests that oxidative injury, similar to that caused by SOD1mutations, plays a pathogenetic role^(18,20,21).

In response to hypoxia, ‘survival’ responses are initiated, includingthe production of stress hormones, erythropoietin, glycolytic enzymesand angiogenic molecules such as VEGF^(22,23). Hypoxia-inducible factors(HIFs) play an essential role in mediating this feedback response viabinding to a defined hypoxia-response element (HRE) in the promotor ofthese genes²³. Hypoxia is a predominant regulator of VEGF expression asinduction of VEGF expression is rapid (minutes), significant (>10-fold)and responsive to minimal changes in oxygen^(22,23). Surprisingly,little attention has been paid to the possible role of hypoxia and HIFsin the initiation of feedback survival mechanisms in the nervous system.While several neurotrophic molecules have been identified^(24,25), fewhave been shown to be regulated by hypoxia. In this regard, it remainsunknown whether hypoxic regulation of VEGF provides neuroprotection,independently of its angiogenic activity.

Further in the nervous system, motor neurons are a well-defined,although heterogeneous group of cells responsible for transmittinginformation from the central nervous system to the locomotor system.Spinal motor neurons are specified by soluble factors produced by,structures adjacent to the primordial spinal cord, signalling throughhomeodomain proteins. Axonal pathfinding is regulated by cell-surfacereceptors that interact with extracellular ligands and once synapticconnections have formed, the survival of the somatic motor neuron isdependent on the provision of target-derived growth factors, althoughnon-target-derived factors, produced by either astrocytes or Schwanncells, are also potentially implicated. Somatic motor neurondegeneration leads to profound disability, and multiple pathogeneticmechanisms including aberrant growth factor signalling, abnormalneurofilament accumulation, excitotoxicity; autoimmunity have beenpostulated to be responsible. Even when specific deficits have beenidentified, for example, mutations of the superoxide dismutase-1 gene infamilial amyotrophic lateral sclerosis and polyglutamine expansion ofthe androgen receptor in spinal and bulbar muscular atrophy, themechanisms by which somatic mortar neuronal degeneration occurs remainunclear. In order to treat motor system degeneration effectively, weneed to understand these mechanisms more thoroughly. Although it hasbeen shown in the art that VEGF has neurotrophic actions on culturedmouse superior cervical ganglia and on dorsal root ganglia (Sondell M.et al. Journal of Neuroscience, (1999) 19, 5731), no studies areavailable about the possible role of VEGF on motor neurons. The presentinvention demonstrates that VEGF has a trophic role for neurons, inparticular motor neurons, and unveils that defective hypoxic regulationof VEGF predisposes to neuron degeneration. Moreover, the presentinvention indicates that VEGF is a therapeutic agent for the treatmentof motor neuron disorders and relates to the usage of polymorphisms inthe VEGF promotor for diagnosing neuron disorders.

AIMS OF THE INVENTION

The present invention aims at providing research tools, diagnostics andtherapeutics in order to improve the health and well-being of patientssuffering from neural disorders. In particular, the present inventionaims at providing the usage of VEGF, or homologues or fragments thereof,in order to treat patients suffering from Alzheimer disease, Parkinson'sdisease, Huntington disease, chronic ischemic brain disease, amyotrophiclateral sclerosis, amyotrophic lateral sclerosis-like diseases and otherdegenerative neuron, in particular motor neuron, disorders. Moreparticularly, the present invention aims at providing the usage ofVEGF₁₆₅ to prevent death of motor neurons in the spinal cord. Thepresent invention also aims at providing receptors, such as neuropilin-1and the vascular endothelial growth factor receptor-2 (VEGFR-2), whichspecifically bind to VEGF and which can be used to screen for othermolecules binding to it. In other words, the present invention aims atproviding therapeutics which stimulate survival of neurons or whichinhibit death of neurons induced by, for example, semaphorin 3A. Thepresent invention further aims at providing an animal which ischaracterized by having an altered (i.e. impaired or non-functional)hypoxia-induced VEGF expression compared to it's wild-type counterpartand which can be used as a research tool to screen for therapeutics asmentioned above. The present invention finally aims at providingpolymorfisms in the VEGF promoter region, such as in the HypoxiaResponsive Element, which can be used to identify individuals prone todevelop a neuron disorder or to treat neuron disorder patients via genetherapy.

FIGURE LEGENDS

FIG. 1: Targeting of the VEGF gene and muscle weakness in VEGF^(m/m)mice. Strategy to delete the HIF-1α binding-element in the VEGFpromoter. The targeting vector pBSK.VEGF^(m), the wild type (VEGF^(WT))VEGF allele, the homologously recombined (VEGF^(neo)) VEGF allele, andthe modified VEGF^(m) allele after Cre-excision of the foxed neocassette are shown. Probes are indicated by solid bars. HRE:hypoxia-response element to which HIF-1alfa binds; the asterisk and “m”denote the HRE deletion.

FIG. 2: Neurotrophic role of VEGF.

A, VEGF₁₆₅, but not VEGF₁₂₁, protects SCN34 motor neurons againstapoptosis (quantified by oligonucleosomes) induced by TNF-alfa (50ng/ml). The survival activity of VEGF₁₆₅ is comparable to that of bFGFor TGF-β1. B, VEGF₁₆₅ also protects SCN34 cells against apoptosisinduced by hypoxia, H₂O₂, or serum deprivation. *: p<0.05 versus 0.01μg/ml VEGF. C, The survival effect of VEGF₁₆₅ (100 ng/ml) is blocked byantibodies (Ab; 50 μg/ml) against VEGFR-2 (R2) and neuropilin-1 (NP1),but not to VEGFR-1 (R1), neuropilin-2 (NP2), or control (ctr) IgG's.Apoptosis was induced by serum starvation (0.5%). None of the antibodiesmodified the baseline level of apoptosis in the absence of VEGF. *:p<0.05 versus control IgG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention shows that deletion of the hypoxia-responseelement in the VEGF promotor effectively abrogates hypoxic induction ofVEGF. Based on the well-known role of VEGF in angiogenesis, it wasanticipated that VEGF^(m/m) mice would suffer impaired VEGF-mediatedangiogenesis. Vascular defects do indeed appear to contribute to thelethality of VEGF^(m/m) embryos but, surprisingly, there are no signs ofvascular insufficiency in surviving VEGF^(m/m) mice under baselineconditions. Furthermore, the neuropathy which is seen in adultVEGF^(m/m) mice is also not due to vascular insufficiency because of thefollowing findings: (i) the number, differentiation and ultrastructureof endothelial cells in the spinal cord, peripheral nerves and musclesof those mice are normal; (ii) endoneural perfusion is normal withoutsigns of leakiness; (iii) pimonidazole staining of spinal cords afterhypoxia is comparable with wt-mice; (iv) infarcts or microangiopathy,typically found in diabetic patients with ischemic neuropathy⁴⁷, areabsent; (v) axonal lesions are not only present at the center (prone toischemia), but also at the periphery of the nerves; (vi) degeneratingmotor neurons lay frequently in the immediate vicinity of normalcapillaries; and (vii) other causes of ischemia including cardiacfailure, anemia, or pulmonary insufficiency are excluded.

The present invention thus relates to a novel transgenic mouse modelwith an impaired hypoxic upregulation of VEGF and characterized byhaving a predisposition to adult onset progressive motor neurondegeneration with neuropathological features, reminiscent of amyotrophiclateral sclerosis. In this novel mouse model the neuropathy is notcaused by vascular defects, but by deprivation of motor neurons from theneurotrophic effect of VEGF. It should be clear however that the presentinvention not only relates to a novel transgenic mouse, but alsoencompasses any non-human transgenic animal (such as a rat, dog, rabbit,non-human primate, etc.) which is characterized by having an impaired ornon-functional hypoxia-induced VEGF expression compared to theirwild-type counterparts. The present invention has significant medicalimplications. First, the genetic etiology of degenerative motor neurondisorders remains undetermined. In less than 2% of ALS cases, mutationsin the SOD1 gene underlie the disease, but the pathogenesis of theremaining 98% remains unknown. Our findings indicate that abnormal generegulation—not function—of VEGF constitutes a novel risk factor formotor neuron degeneration, and compels a search for genetic alterationsthat affect VEGF gene regulation. Even in ALS patients with a SOD1mutation, genetically determined differences in VEGF gene regulation mayexplain the significant intrafamilial phenotypic variability. Second,there is no medical treatment for ALS to date. Our data demonstrate thatVEGF has therapeutic value for motor neuron disorders. The availabilityof an animal model with characteristics of familial ALS (transgenicexpression of mutant SOD1) provides an essential research tool. Ourfindings also indicate that VEGF₁₆₅ protect cortical neurons againstN-methyl-D-aspartate. Third, the present VEGF^(m/m) mouse model of adultonset motor neuron degeneration reflects several clinical andneuropathological features of ALS (progressive muscle atrophy due todegeneration of spinal motor neurons, characterized by neurofilamentinclusions in the perikaryon and axonal swellings^(17-19,32-34)). TheVEGF^(m/m) mouse is therefore a suitable model for evaluation oftherapeutic strategies. Fourth, our data militate for caution againstlong-term use of VEGF-antagonists (currently being tested for treatmentof cancer, diabetes, and rheumatoid arthritis), as they can predisposeto motor neuron degeneration.

The present invention also indicates that VEGF, or homologues, derivatesor fragments thereof, can be used to manufacture a medicament for thetreatment of neuron disorders, and specifically for the treatment ofneuronopathies and more specifically for the treatment of motor neurondisorders and even more specifically for the treatment of amyotrophiclateral sclerosis and amyotrophic lateral sclerosis-like diseases. Inanother embodiment VEGF, or homologues, derivates or fragments thereof,can be used to manufacture a medicament to prevent the death of motorneurons in the spinal cord. In a particular embodiment theVEGF₁₆₅-isoform can be used for the treatment of motor neuron disorders.

By ‘neuron disorders’ it is meant any physiological dysfunction or deathof neurons present in the central nervous system. A non-limited list ofsuch disorders comprises dementia, frontotemporal lobe dementia,Alzheimer's disease, Parkinson's disease, Huntington's disease, priondiseases, neuronopathies and motor neuron disorders. ‘Neuronopathies’are characterized by neuronal cell death of motor neurons or sensoryneurons and hence neuronopathies can be subdivided in motor and sensoryneuron disorders. Motor Neuron Disease (MND) or motor neuron disordersis a group of diseases (disorders) involving the degeneration of theanterior horn cells, nerves in the central nervous system that controlmuscle activity. This leads to gradual weakening and eventually wastingof the musculature (atrophy). Diseases of the motor neuron areclassified according to upper motor neuron (UMN) and/or lower motorneuron (LMN) involvement. Upper motor neurons originate in the brain, inparticular, the motor cortex, and they synapse either directly orindirectly onto lower motor neurons. Upper motor neurons are moreaccurately referred to as pre-motor neurons, and they are responsiblefor conveying descending commands for movement. Lower motor neurons aredivisable into two catergories: visceral and somatic motor neurons.Visceral motor neurons are autonomic pre-ganglionic neurons thatregulate the activity of ganglionic neurons, which innervate glands,blood vessels, and smooth muscle. Somatic motor neurons innervateskeletal muscle and include first, anterior horn cells, which as thename implies, are located in the anterior horn of the spinal cord, andsecond, lower motor neurons located in the cranial nerve nuclei.Amyotrophic lateral sclerosis or ALS is the most frequent form(accounting for around 80% of all cases) of motor neuron disoreders. ALSis known as Lou Gehrig's disease, named after the famous Yankee baseballplayer. The initial symptoms of ALS are weakness in the hands and legsand often fasciculation of the affected muscles. Whichever limbs areaffected first, all four limbs are affected eventually. Damage to theupper motor neurons produces muscle weakness, spasticity and hyperactivedeep tendon reflexes. Lower motor neuron damage produces muscle weaknesswith atrophy, fasciculations, flaccidity and decreased deep tendonreflexes. ALS has features of both upper and lower motor neurons of thecranial nerves, therefore symptoms are isolated to the head and neck.Some patients will also display UMN involvement of the cranial nervesand if this is the sole manifestation it is referred to as Pseudobulbarpulsy. Spinal muscular atrophy or progressive muscular atrophy is a MNDthat does not involve the cranial nerves and is due to lower motorneuron degeneration. Shy-Drager syndrome is characterized by posturalhypotension, incontinence, sweating, muscle rigidity and tremor, and bythe loss of neurones from the thoracic nuclei in the spinal cord fromwhich sympathetic fibres originate. Destructive lesions of the spinalcord result in the loss of anterior horn cells. This is seen inmyelomeningocele and in syringomyelia, in which a large fluid-filledcyst forms in the centre of the cervical spinal cord. Poliomyelitisvirus infection also destroys anterior horn cells. Spinal cord tumoursmay locally damage anterior horn cells either by growth within the cord(gliomas) or by compression of the spinal cord from the outside(meningiomas, schwannomas, metastatic carcinoma, lymphomas).

Dorsal root ganglion cells may be damaged by herpex simplex andvaricella-zoster viruses. Such infections are associated with avesicular rash in the skin regions supplied by those neurones. A similarloss of sensory neurones is observed in ataxia telangiectasia, adisorder associated with progressive cerebellar ataxia and symmetricaltelangiectases of the skin and conjunctiva. Neuronal loss from autonomicganglia is observed in amyloid neuropathies and in diabetes.

A normal number of capillaries developed in VEGF^(m/m) skeletal muscle,but their lumen size was reduced. Irrespective of whether the smallercapillaries were the cause or consequence of the reduced muscle growth,oxygenation was normal and there were no signs of ischemia in VEGF^(m/m)muscle, indicating that perfusion matched the metabolic demands of themuscle fibers. VEGF is able to induce vasodilation which could result instructural vascular remodeling (Laitinen M. et al. (1997) Hum Gene Ther8, 1737) but VEGF levels in normoxic and hypoxic VEGF^(m/m) muscle werenormal. The normal VEGF and reduced IGF-1 levels may suggest that growthof muscle fibers and not of vessels was primarily affected. In contrast,neuronal perfusion was reduced by 50% in VEGF^(m/m) mice, despite anormal number, size and differentiation of the capillaries, and a normalhypercapnic vasoreactive response. Why perfusion is reduced in some butnot in other organs in VEGF^(m/m) mice and whether these organ-specificperfusion deficits relate to the variably reduced baseline and hypoxicVEGF levels in these organs remain to be determined. In contrast toskeletal muscle where the vasculature expands almost 10-fold, theneuronal vascular network expands less but primarily remodels afterbirth (Feher G. et al. (1996) Brain Res Dev Brain Res 91, 209). VEGF hasbeen implicated in the remodeling of the primitive (poorly perfused)capillary plexus at birth to a functionally perfused vasculature in theadult (Ogunshola et al. (2000) Brain Res Dev Brain Res 119, 139). Anintriguing question is therefore whether the reduced neuronal VEGFlevels in VEGF^(m/m) mice reduced neuronal perfusion via impairedvascular remodeling. Irrespective of the mechanism, the neuronalhypoperfusion in VEGF^(m/m) mice might have contributed to the stuntedgrowth and infertility, for instance by impairing secretion ofhypothalamic factors. Mice with hypothalamic or pituitary defects aresmaller and sterile (Chandrashekar V. et al. (1996) Biol Reprod 54,1002). The reduced IGF-1 plasma levels are consistent with suchhypothesis.

While a reduction of neuronal perfusion by 50% did not predisposeVEGF^(m/m) mice to neuronal infarcts, it likely caused chronic neuronalischemia. Animal models of chronic spinal cord ischemia are notavailable, but acute spinal cord ischemia causes significant motorneuron degeneration (Lang-Lazdunski, L. et al. (2000) Stroke 31, 208).Surgically induced cerebral perfusion deficits caused cognitive defectsbut spared rats from motoric dysfunction, and variably caused histologicsigns of neuronal loss (Ohta H. et al (1997) Neuroscience 79, 1039). Ananimal model of spontaneous chronic neuronal ischemia is, however, notavailable. Thus, in a specific embodiment the invention provides a modelfor chronic spinal cord ischemia.

The VEGF^(m/m) mouse model promises to be fruitful for studying theconsequences of neuro-vascular insufficiency on cognitive function andon the progression of neurodegenerative disorders. In a specificembodiment the invention provides a model for cognitive dysfunction andin another specific embodiment the VEGF^(m/m) mouse model is useful tobreed with current mouse models known in the art for neurodegenerativedisorders, for example models for Alzheimer's Disease (Bornemann et al.(2000) Ann NY Acad Sci 908, 260, Van Leuven F. (2000) Prog Neurobiol 61,305, Sommer B. et al. (2000) Rev Neurosci 11, 47).

A diminished nervous blood flow in the brain can lead to brain ischemia.Brain ischemia is a process of delayed neuronal cell death and not aninstantaneous event. A diminished cerebral blood flow initiates a seriesof events (the “ischemic cascade”) that can lead to cell destruction.The goal of neuroprotection is to intervene in the process that ischemicneurons undergo as part of the final common pathway of cell death. Theischemic cascade has been intensively studied, and although it has notbeen completely delineated, certain reproducible aspects are recognized.The normal amount of perfusion to human brain gray matter is 60 to 70mL/100 g of brain tissue/min. When perfusion decreases to <25 mL/100g/min, the neuron is no longer able to maintain aerobic respiration. Themitochondria are forced to switch over to anaerobic respiration, andlarge amounts of lactic acid are generated. This metabolic by-productaccumulates in the extracellular regions and causes a local change inthe pH level. This fundamental change in the environment surroundingischemic cells has been confirmed in humans by magnetic resonancespectroscopy and by single photon emission computed tomography (SPECT).Many studies have focussed on stroke as a model for brain ischemia.However, recently chronic reductions in cerebral blood flow have beenobserved to be associated with aging and progressive neurodegenerativedisorders which can precipitate cognitive failure (Bennet et al. (1998)Neuroreport 9, 161). For example regional cerebral blood flowabnormalities to the frontal and temporal regions are observed indepressed patients with cognitive impairment (Dolan et al. (1992) JNeurol Neurosurg Psychiatry 9, 768, Ritchie et al. (1999) Age Ageing 28,385). In Alzheimer's disease (AD), an example of a neurodegenerativedisorder, an impaired cerebral perfusion originates in themicrovasculature which affects the optimal delivery of glucose andoxygen and results in a breakdown of metabolic pathways in brain cellssuch as in the biosynthetic and synaptic pathways. It is proposed thattwo factors need to be present before cognitive dysfunction andneurodegeneration is expressed in AD brain, advanced aging, and thepresence of a specific condition that further lowers cerebral perfusion(de la Torre (1999) Acta Neuropathol 98, 1). Further in AD a criticalthreshold cerebral hypoperfusion is a self-perpetuating, contained andprogressive circulatory insufficiency that will destabilize neurons,synapses, neurotransmission and cognitive function, creating in its wakea neurodegenerative process characterized. by the formation of senileplaques, neurofibrillary tangles and amyloid angiopathy.

Cognition is referred to the process involved in knowing, or the act ofknowing, which in its completeness includes perception and judgement.Cognition includes every mental process that can be described as anexperience of knowing as distinguished from an experience of feeling orof willing. It includes, in short, all processes of consciousness bywhich knowledge is built up, including perceiving, recognizing,conceiving, and reasoning. The essence of cognition is judgement, inwhich a certain object is distinguished from other objects and ischaracterized by some concept or concepts. Cognitive disorders orcognitive dysfunction are disturbances in the mental process related tocognition. An overview of cognitive disorders (also called amnesticdisorders) can be found in the Diagnostic and Statistical Manual ofMental Disorders (DSM-IV™) (ISBN 0890420629).

In a specific embodiment the novel VEGF^(m/m) mouse model can be used toidentify and/or to test molecules to prevent and/or to treat neuronalischemia or neurodegenerative disorders and/or cognitive dysfunction.

In another embodiment the present invention further indicates that VEGF,or homologues, derivatives or fragments thereof, can be used for themanufacture or a medicament to prevent and/or to treat neuronal ischemiasuch as brain ischemia. And in yet another embodiment VEGF, orhomologues, derivatives or fragments thereof, can be used for themanufacture or a medicament to prevent and/or to treat cognitivedysfunction.

VEGF and homologues such as VEGF-B, VEGF-C, VEGF-D and PLGF aredescribed in detail in Neufeld G. et al, Faseb Journal, 13, 9-22, 1999,Korpelainen E. I. et al, Curr. Opin. Cell. Biol. 10, 159-164, 1998 andin Joukov, V. et al. J. Cell. Physiol. 173, 211-215, 1997. Inparticular, certain of the VEGF genes, homologues, fragments, andderivatives thereof that are useful for practicing the claimed inventionare described in GenBank Accession Nos. NM 003376 (“Homo sapiensvascular endothelial growth factor (VEGF) mRNA”); NM 003377 (“Homosapiens vascular endothelial growth factor B (VEGFB) mRNA”); NM 005429(“Homo sapiens vascular endothelial growth factor C (VEGFC) mRNA”); NM004469 (“Homo sapiens c-fos induced growth factor (vascular endothelialgrowth factor D) (FIGF) mRNA); AF 024710 (“Homo sapiens vascular growthfactor (VEGF₁₆₅)) mRNA, 3′UTR, mRNA sequence”); and U.S. Pat. Nos.6,013,780 (“VEGF₁₄₅ expression vectors”); 5,935,820 (“Polynucleotidesencoding vascular endothelial growth factor 2”); 5,607,918 (“Vascularendothelial growth factor-B and DNA coding therefore”). The preferrednucleic acids of the invention encode the above-mentioned angiogenicgrowth factor polypeptides, as well as their homologues and alleles andfunctionally equivalent fragments or variants of the foregoing. Forexample, human VEGF 1 (VEGF A) exists in four principal isoforms,phVEGF₁₂₁; phVEGF₁₄₅; phVEGF₁₆₅; and phVEGF₁₈₉. Preferably, the VEGFnucleic acid has the nucleotide sequence encoding an intact humanangiogenic growth factor polypeptide, i.e., the complete coding sequenceof the gene encoding a human VEGF; however the invention also embracesthe use of nucleic acids encoding fragments of an intact VEGF.

Homologues and alleles of the nucleic acid and amino acid sequencesreported for the VEGF genes, such as those mentioned herein, also arealso within the scope of the present invention. In addition, nucleicacids of the invention include nucleic acids which code for the VEGFpolypeptides having the sequences reported in the public databasesand/or literature, but which differ from the naturally occurring nucleicacid sequences in codon sequence due to the degeneracy of the geneticcode. The invention also embraces isolated functionally equivalentfragments, variants, and analogs of the foregoing nucleic acids;proteins and peptides coded for by any of the foregoing nucleic acids;and complements of the foregoing nucleic acids. ‘Functionally’ meansthat the fragments, variants and analogs must have the capacity to treata neuron disorder and in particular a motor neuron disorder.

The term ‘derivatives’ refers to any variant, mutant or peptidecomposition of VEGF, which retains the capacity, or can be used, totreat degenerative motor neuron disorders as defined above. The latterterm also includes post-translational modifications of the amino acidsequences of VEGF such as glycosylation, acetylation, phosphorylation,modifications with fatty acids and the like. Included within thedefinition are, for example, amino acid sequences containing one or moreanalogues of an amino acid (including unnatural amino acids), amino acidsequences with substituted linkages, peptides containing disulfide bondsbetween cysteine residues, biotinylated amino acid sequences as well asother modifications known in the art. The term thus includes any proteinor peptide having an amino acid residue sequence substantially identicalto a sequence specifically shown herein in which one or more residueshave been conservatively substituted with a biologically equivalentresidue. Examples of conservative substitutions include the substitutionof one-polar (hydrophobic) residue such as isoleucine, valine, leucineor methionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagines, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another. The phrase “conservative substitution” alsoincludes the use of a chemically derivatized residue in place of anon-derivatized residue provided that the resulting protein or peptideis biologically equivalent to the protein or peptide of the invention.‘Chemical derivative’ refers to a protein or peptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules, include but are not limited to,those molecules in which free amino groups have been derivatized to formamine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloracetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-imbenzylhistidine.Also included as chemical derivatives are those proteins or peptides,which contain one or more naturally-occurring amino acid derivatives ofthe twenty standard amino acids. For example: 4-hydroxyproline may besubstituted for praline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine. Theproteins or peptides of the present invention also include any proteinor peptide having one or more additions and/or deletions or residuesrelative to the sequence of a peptide whose sequence is shown herein, solong as the peptide is biologically equivalent to the proteins orpeptides of the invention. When percentage of sequence identity is usedin reference to polypeptides (i.e. homologues), it is recognized thatresidue positions which are not identical often differ by conservativeaa substitutions, where aa residues are substituted for other aaresidues with similar chemical properties (for example charge orhydrophobicity) and therefore do not change the functional properties ofthe polypeptide. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well known to those skilled in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than asfull mismatch, thereby increasing the percentage sequence identity.Thus, for example (and as described in WO 97/31116 to Rybak et al),where an identical aa is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. In this regard, it should be clearthat polypeptides, or parts thereof, comprising an aa sequence with atleast 55%, preferably 75%, more preferably 85% or most preferably 90%sequence identity with the amino acid sequence of VEGF, or partsthereof, fall within the scope of the present invention. It should alsobe clear that polypeptides which are immunologically reactive withantibodies raised against VEGF, or parts thereof, fall within the scopeof the present invention.

The term ‘fragments of VEGF’ refers to any fragment, including anymodification of said fragment as described above, which retains thecapacity, or can be used, to treat neuron disorders and in particularmotor neuron disorders.

The terms ‘pharmaceutical composition’ or ‘medicament’ or ‘use for themanufacture of a medicament to treat’ relate to a composition comprisingVEGF or homologues, derivatives or fragments thereof as described aboveand a pharmaceutically acceptable carrier or excipient (both terms canbe used interchangeably) to treat diseases as indicated above. Suitablecarriers or excipients known to the skilled man are saline, Ringer'ssolution, dextrose solution, Hank's solution, fixed oils, ethyl oleate,5% dextrose in saline, substances that enhance isotonicity and chemicalstability, buffers and preservatives. Other suitable carriers includeany carrier that does not itself induce the production of antibodiesharmful to the individual receiving the composition such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids and amino acid copolymers. The ‘medicament’ may be administered byany suitable method within the knowledge of the skilled man. Thepreferred route of administration is parenterally. In parentaladministration, the medicament of this invention will be formulated in aunit dosage injectable form such as a solution, suspension or emulsion,in association with the pharmaceutically acceptable excipients asdefined above. However, the dosage and mode of administration willdepend on the individual. Generally, the medicament is administered sothat the protein, polypeptide, peptide of the present invention is givenat a dose between 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kgand 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably, it isgiven as a bolus dose. Continuous infusion may also be used and includescontinuous subcutaneous delivery via an osmotic minipump. If so, themedicament may be infused at a dose between 5 and 20 μg/kg/minute, morepreferably between 7 and 15 μg/kg/minute.

In a particularly preferred embodiment the infusion with a compositioncomprising VEGF or homologues, derivatives or fragments thereof isintrathecal. Intrathecal administration can for example be performed bymeans of surgically implanting a pump and running a catheter to thespine. It should be mentioned that intrathecal administration of VEGF orhomologues, derivatives or fragments thereof is a particularly importantaspect of the present invention. Indeed, since we have shown that VEGFhas a neurotrophic aspect on neurons and more particularly on motorneurons, intrathecal administration is a preferred way. This is incontrast with WO 0062798 were therapeutic angiogenesis is aimed at inorder to treat ischemic peripheral neuropathy.

Instead of delivering VEGF or a homologue, derivative or fragmentthereof, as a protein to a patient in need for treatment of a neurondisorder or more particularly a motor neuron disorder, also a nucleicacid encoding VEGF, or a homologue, derivative or fragment thereof canbe delivered to said patient. In that case the nucleic acid encodingVEGF or homologue, derivative or fragment thereof, can be operativelycoupled to a promoter that can express said angiogenic growth factor ina target cell (e.g., an endothelial cell, a nerve cell, a muscle cell, aneuron, a motor neuron). Often the nucleic acid is contained in anappropriate expression vector (e.g., plasmid, adenoviral vector,modified adenoviral vector, retroviral vector, liposome) to moreefficiently genetically modify the target cell and achieve expression ofsaid angiogenic growth factor. For example, in WO 9831395 the selectivetransfer of genes into motor neurons is fully described.

In another embodiment of the invention it is shown that the VEGF₁₆₅isoform, but not the VEGF₁₂₁ isoform, provides neuroprotection viabinding to neuropilin-1 and VEGFR-2.

In yet another embodiment of the invention inhibitors of Sema3A, amolecule which is implicated in neuronal apoptosis⁴³ and axonretraction⁴⁴, and inhibits binding of VEGF₁₆₅ to neuropilin-1⁹, can bemade and used for the treatment of neuron disorders. Neuropilin-1 alsobinds Sema3A, implicated in repulsion of motor projections duringdevelopment¹¹⁻¹⁵. Neuropilin-1 and Sema3A are expressed in the ventralhorn after birth, but their role has remained enigmatic. A recent invitro study suggested a role for Sema3A in apoptosis of sympathetic andcerebellar neurons⁴³, whereas downregulation of Sema3A was suggested tobe a prerequisite for axonal. regeneration after nerve injury⁴⁴.

In yet another embodiment VEGF, or homologues, derivatives or fragmentsthereof can be administrated for the prevention of neuronal loss or morespecifically of motor neuronal loss in the spinal cord in for examplesurgical indications where an ischemic insult to neurons or motorneurons can be expected. The initiation of neuroprotective pathwaysduring hypoxia is required, as these vital post-mitotic motor neuronscannot regenerate after a lethal hypoxic insult. In this regard only afew neuroprotective molecules such as NGF, bFGF, TGFβ1⁵²⁻⁵⁴ have beencharacterized. The present invention clearly indicates that VEGF is apotent neuroprotective agent, as regulation of its expression by hypoxiais rapid (minutes), significant (>10-fold) and sensitive to smallchanges in oxygen. The absence of neuroprotective VEGF responses inVEGF^(m/m) mice—even though they might only occur transiently, butrepetetively—may explain why motor neurons in these mice ultimatelydegenerate after cumulative sublethal mini-insults of hypoxia.

Neuropilin-1 (NP-1), a receptor for the VEGF₁₆₅ isoform^(9,10) and forthe neurorepellant semaphorin 3A (Sema3A)¹¹⁻¹³ is shown to be essentialfor guiding neuronal projections during embryonic patterning¹¹⁻¹⁵.However, it is not known if NP-1 and/or Sema3A have any role in neuronalfunction after birth. In a further embodiment the invention furtherprovides methods for identifying compounds or molecules which bind onthe neuropilin receptor and VEGFR-2 and stimulate the survival ofneurons and more particularly motor neurons. In this invention theresults show that VEGF₁₆₅, via binding to neuropilin-1 and VEGFR-2,mediates survival of NSC34 motor neurons. Both receptors are expressedon motor neurons in adult spinal cords in vivo, and are thereforeaccessible to VEGF₁₆₅, produced by the motor neuron itself or by othernearby cells. Neuropilin-1 and VEGFR-2 act as co-receptors instimulating endothelial cell motility^(9,10) and also cooperate inmediating neuronal survival. These methods are also referred to as ‘drugscreening assays’ or ‘bioassays’ and typically include the step ofscreening a candidate/test compound or agent for the ability to interactwith (e.g. bind to) neuropilin-1 and VEGFR-2. Candidate compounds oragents, which have this ability, can be used as drugs to treatdegenerative disorders. Candidate/test compounds such as smallmolecules, e.g. small organic molecules, and other drug candidates canbe obtained, for example, from combinatorial and natural productlibraries.

The invention also provides methods for identifying compounds or agentswhich can be used to treat degenerative neurons. These methods are alsoreferred to as ‘drug screening assays’ or ‘bioassays’ and typicallyinclude the step of screening a candidate/test compound or agent for theability to interact with (e.g., bind to) neuropilin-1 and VEGFR-2.Candidate/test compounds or agents which have this ability, can be usedas drugs to treat degenerative neuron disorders. Candidate/testcompounds such as small molecules, e.g., small organic molecules, andother drug candidates can be obtained, for example, from combinatorialand natural product libraries. In one embodiment, the invention providesassays for screening candidate/test compounds which interact with (e.g.,bind to) neuropilin-1 and VEGFR-2. Typically, the assays are cell-freeassays which include the steps of combining neuropilin-1 and VEGFR-2 anda candidate/test compound, e.g., under conditions which allow forinteraction of (e.g. binding of) the candidate/test compound withneuropilin-1 and VEGFR-2 to form a complex, and detecting the formationof a complex, in which the ability of the candidate compound to interactwith neuropilin-1 and VEGFR-2 is indicated by the presence of thecandidate compound in the complex. Formation of complexes between theneuropilin-1 and the candidate compound can be quantitated, for example,using standard immunoassays. The neuropilin-1 employed in such a testmay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. In another embodiment, theinvention provides screening assays to identify candidate/test compoundswhich stimulate neuropilin-1 and VEGFR-2 or inhibit binding of sema3A toneuropilin-1 and/or VEGFR-2. Typically, the assays are cell-free assayswhich include the steps of combining neuropilin-1 and VEGFR-2 of thepresent invention or fragments thereof, and a candidate/test compound,e.g., under conditions wherein but for the presence of the candidatecompound, the neuropilin-1 and VEGFR-2 or a biologically active portionthereof interacts with (e.g., binds to) the target molecule or theantibody, and detecting the formation of a complex which includes theneuropilin-1 and the target molecule or the antibody, or detecting theinteraction/reaction of neuropilin-1 and the target molecule orantibody. Detection of complex formation can include direct quantitationof the complex.

To perform the above described drug screening assays, it is feasible toimmobilize neuropilin-1 and VEGFR-2 or its (their) target molecule(s) tofacilitate separation of complexes from uncomplexed forms of one or bothof the proteins, as well, as to accommodate automation of the assay.Interaction (e.g., binding of) of neuropilin-1 and VEGFR-2 to a targetmolecule can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtitre plates, testtubes, and microcentrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows the protein to be boundto a matrix. For example, neuropilin-1-His tagged can be adsorbed ontoNi-NTA microtitre plates (Paborsky et al., 1996), or neuropilin-1-ProtAfusions adsorbed to IgG, which are then combined with the cell lysates(e.g., ³⁵S-labeled) and the candidate compound, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, theplates are washed to remove any unbound label, and the matriximmobilized and radiolabel determined directly, or in the supernatantafter the complexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofneuropilin-1 binding protein found in the bead fraction quantitated fromthe gel using standard electrophoretic techniques. Other techniques forimmobilizing protein on matrices can also be used in the drug screeningassays of the invention. For example, either neuropilin-1 and VEGFR-2 orits target molecules can be immobilized utilizing conjugation of biotinand streptavidin. Biotinylated neuropilin-1 and VEGFR-2 can be preparedfrom biotin-NHS(N-hydroxy-succinimide) using techniques well known inthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with neuropilin-1 butwhich do not interfere with binding of the protein to its targetmolecule can be derivatized to the wells of the plate, and neuropilin-1and VEGFR-2 trapped in the wells by antibody conjugation. As describedabove, preparations of a neuropilin-1-binding protein and a candidatecompound are incubated in the neuropilin-1-presenting wells of theplate, and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the neuropilin-1 targetmolecule and VEGFR-2-target molecule, or which are reactive withneuropilin-1 and VEGFR-2 and compete with the target molecule; as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the target molecule. Another technique for drugscreening which provides for high throughput screening of compoundshaving suitable binding affinity to neuropilin-1 and VEGFR-2 isdescribed in detail in, “Determination of Amino Acid SequenceAntigenicity” by Geysen HN, WO 84/03564, published on 13/09/84. Insummary, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The protein test compounds are reacted with fragments ofneuropilin-1 or/and VEGFR-2 and washed. Bound neuropilin-1 is thendetected by methods well known in the art. Purified neuropilin-1 or/andVEGFR-2 can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support. This invention also contemplates theuse of competitive drug screening assays in which neutralizingantibodies capable of binding neuropilin-1 or/and VEGFR-2 specificallycompete with a test compound for binding neuropilin-1 or/and VEGFR-2. Inthis manner, the antibodies can be used to detect the presence of anyprotein, which shares one or more antigenic determinants withneuropilin-1 and VEGFR-2

No genetic mutations of the VEGF gene, resulting in gene disruption,have thus far been linked to human disease, likely because absence ofeven a single VEGF allele is embryonically lethal^(1,2,26). Recently,however, impaired hypoxic regulation of VEGF has been shown toconstitute a risk factor for ischemic heart disease²⁷. Whether thisabnormal VEGF gene regulation—not function—may predispose topathological disorders is, however, not known. In another embodiment ofthe invention polymorphisms in the regulatory region of the VEGF gene,which have an influence on the hypoxic regulation of said gene, can bedetected and used diagnostically to identify patients at risk to developa neuropathy or more specifically a motor neuropathy when exposed tobrief periods of ischemia. Hypoxia-induced transcription of VEGF mRNA ismediated, at least in part, by the binding of hypoxia-inducible factor 1(HIF-1) to an HIF-1 binding site located in the VEGF promotor. By thedetection of polymorphisms that influence the hypoxic regulation of theVEGF gene it is also meant polymorphisms in the HIF-1 transcriptionfactor, additional HIF-1-like factors, upstream regulators of HIF-1 andHIF-1-like transcription factors comprising the oxygen-sensor,additional factors binding to the 5′ and 3′ untranslated region of theVEGF mRNA and in the internal ribosomal entry site present in the 5′untranslated region of VEGF (Neufeld G. et al. FASEB J. 13, 9-22, 1999).

Several procedures have been developed for scanning genes in order todetect polymorphisms in genes. In terms of current use, many of themethods to scan or screen genes employ slab or capillary gelelectrophoresis for the separation and detection step in the assays.Some of these methods comprise Single strand conformational polymorphism(SSCP) (Orita et al., “Detection of Polymorphisms of Human DNA by GelElectrophoresis as Single-Stranded Conformation Polymorphisms,” Proc.Natl. Acad. Sci. USA 86, 2766 (1989)), denaturing gradient gelelectrophoresis (DGGE) (Abrams et al., “Comprehensive Detection ofSingle Base Changes in Human Genomic DNA Using Denaturing Gradient GelElectrophoresis and a GC Clamp,” Genomics 7, 463 (1990)), chemicalcleavage at mismatch (CCM) (J. A. Saleeba & R. G. H. Cotton, “ChemicalCleavage of Mismatch to Detect Mutations,” Methods in Enzymology 217,286 (1993)), enzymatic mismatch cleavage (EMC) (R. Youil et al,“Screening for Mutations by Enzyme Mismatch Cleavage with T4Endonuclease VII,” Proc. Natl. Acad. Sci. USA 92, 87 (1995)), and“cleavase” fragment length polymorphism (CFLP). Still other methodsfocus on the use of mass spectrometry as a genetic analysis tool. Massspectrometry requires minute samples, provides extremely detailedinformation about the molecules being analyzed including high massaccuracy, and is easily automated. U.S. Pat. No. 5,965,363 describesnucleic acid analysis by means of mass spectrometric analysis.

In another embodiment of the invention the Hypoxia Response Element(HRE) can be used for the treatment of neuron disorders or morespecifically motor neuron disorders. VEGF, or homologous, derivates orfragments thereof, can be placed under hypoxic control by splicing saidgenes to one or more HRE elements. These constructs can then be used ingene therapy.

Gene therapy means the treatment by the delivery of therapeutic nucleicacids to patient's cells. This is extensively reviewed in Lever andGoodfellow 1995; Br. Med. Bull., 51, 1-242; Culver 1995; Ledley, F. D.1995. Hum. Gene Ther. 6, 1129. To achieve gene therapy there must be amethod of delivering genes to the patient's cells and additional methodsto ensure the effective production of any therapeutic genes. There aretwo general approaches to achieve gene delivery; these are non-viraldelivery and virus-mediated gene delivery. The best characterizedvirus-mediated gene delivery system uses replication defectiveretroviruses to stably introduce genes into patients' cells.

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and cannot be construed as to restrict the invention in anyway.

EXAMPLES 1. Targeted Deletion of the HIF-Binding Site in the VEGFPromotor

Targeted deletion in the VEGF promotor of the hypoxia-response element(HRE), i.e. the binding site for the hypoxia-inducible factors (HIF)²³,was achieved using CrelloxP-mediated targeting (FIG. 1), and confirmedby Southern blot analysis. Impaired hypoxic induction of VEGF inembryonic stem cells, homozygous for the HRE-deletion (VEGF^(m/m)), wasconfirmed by Northern blot analysis and by measurements of VEGF releaseduring 36 h hypoxia (19±5 pg/ml after normoxia versus 45±6 pg/ml afterhypoxia in VEGF^(+/+) cells, n=6, p<0.05; 11±2 pg/ml after normoxiaversus 13±3 μp/ml after hypoxia in VEGF^(m/m) cells, n=6; p=NS).Deletion of the HIF-binding site in the VEGF gene was as effective asdeletion of the HIF-1α gene itself²⁸ in abolishing hypoxic upregulationof VEGF (13±4 pg/ml after normoxia versus 14±2 pg/ml after hypoxia inHIF-1α^(−/−) cells; n=6; p=NS). VEGF^(m/m) embryos were recovered at anormal Mendelian frequency. Of the VEGF^(m/m) mice, 30% died beforebirth and another 30% within the first postnatal days, while theremaining 40% survived more than 12 months. Here, the phenotype of thesurviving VEGF^(m/m) mice is described; the embryonic and neonatalphenotypes will be reported separately.

2. Motor Coordination and Muscular Performance in VEGF^(m/m) Mice

VEGF^(m/m) mice appeared normal until 4 months, but thereafter developedsymptoms of motor neuron disease. They became progressively less mobile,and exhibited signs of severe muscle weakness and limb paresis. Beyondsix months of age, mutant mice were too weak to turn over when placed ontheir back, slapped their feet while walking, and had a waddling gaitand scoliosis. When lifted by their tails, they reflexively contractedtheir limbs to the trunk and remained immobile, whereas wild type miceextended their limbs and struggled. VEGF^(m/m) mice developed a coarsefur suggestive of impaired grooming and appeared thin along their flanksand legs: Notably, when asymptomatic two months old VEGF^(m/m) mice werekept in a hypoxic chamber (10% O₂), they developed neurological signs(difficulty in turning over, reflex contracture when lifted by tail)within two weeks, indicating that hypoxia markedly accelerated the onsetand progression of the phenotype. Beyond 4 to 6 months of age,VEGF^(m/m) mice performed significantly less well than wild typelittermates in a number of motor coordination and muscle performancetests²⁹, including the treadmill-wheel test, grid test, rotating axletest and the footprint test (distance between the central pads of thehindfeet: 65±5 mm for VEGF^(+/+) mice versus 45±5 mm for VEGF^(m/m)mice; n=7; p<0.05). Compared to VEGF^(+/+) mice, VEGF^(m/m) mice weresignificantly less active at night (number of treadmill-wheel turns:5400±600 for VEGF^(+/+) mice versus 2700±400 for VEGF^(m/m) mice; n=7;p<0.05) and for much shorter periods (minutes of intense activity:150±40 for VEGF^(+/+) mice versus 14±6 for VEGF^(m/m); n=7; p<0.05). Inthe ‘grid’ test (mice are placed on a grid, that is subsequently turnedupside-down), five of seven VEGF^(+/+) mice hung on to the grid for atleast one minute. Two VEGF^(+/+) mice moved so actively that theydropped from the rack after 23 and 45 seconds. In contrast, four of six20 week-old VEGF^(m/m) mice fell already off the grid after 8 seconds,and only two mutant mice managed to hold on to the grid by not moving atall. When testing their grip strength (mice are forced to hang withtheir forelimbs on a horizontal thread), all VEGF^(+/+) mice (n=7)immediately grabbed the thread with their hindlimbs. In contrast,VEGF^(m/m) mice had difficulties in grabbing the thread with theirhindlimbs, hung immobile and sagged. When they finally succeeded (fiveof six mice), VEGF^(m/m) mice could not hold on to the thread and felloff. VEGF^(m/m) mice also performed worse in the ‘rotating axle’ test,used to evaluate how long mice could stay on a rotating axle beforefalling off: all but one VEGF^(+/+) mice (n=8) stayed on the axle for atleast two minutes (time of analysis), whereas all of six VEGF^(m/m) micefell off after less than a minute (53±20 sec). Pain threshold, a sensoryfunction measured as the paw-lick response in a hot plate test²⁹ wasnormal in VEGF^(m/m) mice (the time to lick the front or rear paws forboth genotypes was 7±1 s and 10±2 s, respectively; n=6; p=NS). However,VEGF^(+/+) mice jumped out of the box after 100±20 s, whereas VEGF^(m/m)mice were too weak to escape during this period.

3. Muscle Atrophy in VEGF^(m/m) Mice

Skeletal muscles in VEGF^(m/m) mice beyond 4 months of age showed signsof neurogenic atrophy. The wet weight of the plantar and dorsal flexormuscles was 170±14 mg and 92±19 mg in VEGF^(+/+) mice versus 94±5 mg and58±18 mg in VEGF^(m/m) mice (n=3; p<0.05). Initially, a variable numberof fibers were atrophic, but in older animals, most muscle fibers wereseverely atrophic, “angulated” or “elongated”, characteristics ofdenervated fibers. Muscle fiber size was decreased by more than 50% inVEGF^(m/m) mice (cross-sectional area: 1700±200 μm² in VEGF^(+/+) miceversus 700±100 μm² in VEGF^(m/m) mice; n=8; p<0.05). Regenerating musclefibers, identified by their centrally located vesicular nucleus, smallersize and desmin-immunoreactivity, were commonly observed in VEGF^(m/m)mice. Myosin ATPase staining revealed atrophy of all fiber types(type-I, -IIa, and -IIb). In contrast to the typical chessboard patternof all fiber types in VEGF^(+/+) mice, grouping of fibers of a similartype, a sign of reinnervation, was observed in VEGF^(m/m) mice. Musclespindles—involved in reflex control—were present in both genotypes(number of spindles/quadriceps section: 3.9±0.9 in VEGF^(+/+) miceversus 4.5±0.8 in VEGF^(m/m) mice; n=5; p=NS). Myopathic changes(sarcolemma desintegration, fiber necrosis, loss of muscle fibers,elevated plasma creatine kinase levels or fibrosis) were not detected inVEGF^(m/m) mice. The muscle atrophy in VEGF^(m/m) mice did not resemblethe degenerative features of primary myopathies. Indeed, there was noloss of muscle fibers and, because of shrinkage, the density of themuscle fibers was increased in VEGF^(m/m) mice (1250±190 cells/mm²) ascompared to VEGF^(+/+) mice (720±80 cells/mm²; p<0.05). Unlike inmyopathies, there were no signs of fiber necrosis, sarcomere lysis orsarcolemma disruption (ultrastructural analysis; normal titin and desminstaining; absence of intracellular albumin), fatty infiltration,fibrosis (sirius red staining), or dystrophic calcification. Plasmalevels of creatine kinase (released upon myocyte death) were normal in 8month-old VEGF^(m/m) mice (88±20 U/ml in VEGF^(+/+) mice versus 94±9U/ml in VEGF^(m/m) mice; n=5; p=NS). In addition, atrophy was confinedto skeletal and not to cardiac muscle, and was not caused by systemicdisorders. Atrophy was confined to skeletal muscle fibers, sincecardiomyocytes were not affected (cross-sectional area: 130±10 μm² inVEGF^(+/+) mice versus 125±8 μm² in VEGF^(m/m) mice; n=5; p=NS).Structural changes in muscle fibers were also not due to infectiousdisease (pathogen-free health report), inflammatory disorders ofconnective tissue or blood vessels (no signs of vasculitis), metabolicdisorders (normal plasma glucose levels), or storage abnormalities inglycogen or lipids.

4. Motor Neuron Degeneration in VEGF^(m/m) Mice

Evidence for a neurodegenerative process was obtained by analysis of thespinal cord and peripheral nerves. Nissl staining revealed a comparablenumber of neurons in the ventral horn in the spinal cord at young age(12 weeks) in both genotypes (neurons with a clearly identifiablecytoplasm/ventral horn section: 110±2 in VEGF^(+/+) mice versus 107±6 inVEGF^(m/m) mice; n=3; p=NS), indicating that deletion of the HIF-bindingsite in the VEGF promotor per se did not cause abnormal neuronaldevelopment. However, beyond 7 months of age, fewer neurons weredetected in the ventral horn in VEGF^(m/m) mice (neurons/ventral hornsection: 110±3 in VEGF^(+/+) mice versus 98±4 in VEGF^(m/m) mice; n=8;p<0.05). Immunostaining for choline acetyltransferase (ChAT), a markerof motor neurons, revealed 30% fewer motor neurons in VEGF^(m/m) than inVEGF^(+/+) mice (ChAT-positive neurons/spinal cord section: 26±2 inVEGF^(+/+) mice versus 18±2 in VEGF^(m/m) mice; n=4; p=0.05). Incontrast to VEGF^(+/+) mice, the neuronal cell bodies (perikarya) andproximal axons of motor neurons in VEGF^(m/m) mice contained inclusionsof phosphorylated neurofilament (NF_(P)), a hallmark of motor neurondisease³¹ (number of NF_(P)-positive neurons/spinal cord section: nonein VEGF^(+/+) mice versus 7±2 in VEGF^(m/m) mice; n=6; p<0.05). TheseNF_(P)-positive motor neurons were smaller in size (250±20 μm²) thanNF_(P)-negative motor neurons in VEGF^(+/+) mice (500±40 μm²; n=4,p<0.05). The complexity and occurrence of dephosphorylated neurofilament(NF)-positive axons and of MAP-2-positive dendrites was comparable inboth genotypes, even though more neurons, in VEGF^(m/m) mice tended toaccumulate dephosphorylated NF in their perikaryon. Focal axon swellings(‘spheroids’), also found in ALS patients^(33,34), occurred in thespinal cord in VEGF^(m/m) but not in VEGF^(+/+) mice (number of swollenaxons/spinal cord section at 31 weeks of age: none in VEGF^(+/+) miceversus 17±1 in VEGF^(m/m) mice; n=7). Swollen axons with dense axoplasmwere primarily located in the ventral horn, whereas the dorsal spinalcord or the corticospinal tracts appeared relatively spared. A fractionof these swollen axons was immunoreactive for synaptophysin, a sign ofimpaired axonal transport, and for ubiquitin, which binds damagedproteins in neurodegenerative conditions such as ALS³⁵. They oftencontained neurofilament inclusions, as revealed by Bielschowski stainingand immunostaining for phosphorylated neurofilament (NF_(P)). Comparedto VEGF^(+/+) mice, a prominent reactive astrocytosis was consistentlyobserved in the spinal cord of VEGF^(m/m) mice, but characteristicallyonly in the ventral horn. Numerous hypertrophic astrocytes accumulatedin the ventral and intermediate zones (GFAP-positive area in graymatter: 0.8±0.4% in VEGF^(+/+) mice versus 7±1% in VEGF^(m/m) mice; n=4;p<0.05), and in the ventral white matter (GFAP-positive area in whitematter: 8±3% in VEGF^(+/+) mice versus 31±2% in VEGF^(m/m) mice; n=4;p<0.05).

5. Axon Degeneration in VEGF^(m/m) Mice

Signs of Wallerian degeneration and significant loss of large axons werefound. Some fibers were completely replaced by the more numerousactivated macrophages, phagocytosing disrupted myelin sheets (number ofF4/80-positive cells/mm²: 150±27 in VEGF+/+ mice versus 340±20 inVEGF^(m/m) mice; n=6; p<0.05). Endoneural fibrosis and expression ofGFAP, a marker of denervated Schwann cells, were more prominent inmutant nerves.

6. Electrophysiology of VEGF^(m/m) Mice

Electromyographic (EMG) recordings during rest and muscle contractionrevealed clear signs of denervation and reinnervation. Diffusespontaneous activity (fibrillation potentials, isolated or salvo's ofpositive sharp waves), together with polyphasic motor unit actionpotentials (MUAP's) of normal amplitude, and unstable satellitepotentials were observed in the superficial gastrocnemius muscle, theparavertebral muscles and the diaphragm in VEGF^(m/m) but not inVEGF^(+/+) mice. In the diaphragm, denervation was evidenced by areduced recruitment of MUAP's during inspiration in VEGF^(m/m) mice(number of MUAP's per inspiratory burst: 124±20 in VEGF^(+/+) miceversus 56±4 in VEGF^(m/m) mice; n=7; p<0.05). Compared to the normal bi-or triphasic pattern of MUAPs in VEGF^(+/+) mice, long-durationpolyphasic MUAP's were regularly detected. The duration of inspiratorybursts in VEGF^(m/m) mice remained normal (2300±230 ms in VEGF^(+/+)mice versus 2100±300 ms in VEGF^(m/m) mice; n=7; p=NS), and theamplitude of the largest diaphragmatic MUAP's were preserved inVEGF^(m/m) mice (MUAPs with an amplitude >200 μV per inspiratory burst:14±2 in VEGF^(+/+) mice versus 11±4 in VEGF^(m/m) mice; n=7; p=NS),consistent with an ongoing process of denervation and reinnervation.Furthermore, in contrast to the innervation of individual endplates by asingle terminal axon, terminal axons in VEGF^(m/m) mice more oftenbranched as thin (presumably unmyelinated) sprouts to two or moreendplates. Latencies of compound muscle action potentials (measured fromsite of stimulation to site of recording) were somewhat increased inmutant mice (810±36 μs in VEGF^(+/+) mice versus 1030±44 μs inVEGF^(m/m) mice; n=7; p<0.05), compatible with the axonal loss. Sensorynerve function appeared electrophysiologically normal: sensory nerveaction potential (SNAP) amplitudes were 100±7 μV in VEGF^(+/+) miceversus 120±14 μV in VEGF^(m/m) mice (n=5; p=NS), and sensory nerveconduction velocities were 33±2 m/s in VEGF^(+/+) mice versus 30±2 m/sin VEGF^(m/m) mice (n=5; p=NS). These findings are consistent with apurely motor neurogenic disorder.

7. Normal Vascular Growth in VEGF^(m/m) Mice

Because of the well-known angiogenic role of VEGF, VEGF^(m/m) mice wereexamined for vascular defects. However, there were no signs of vascularinsufficiency in the skeletal muscle, peripheral nerves, or spinal cord.(i) In muscle, because of the atrophy, capillary densities were higherin VEGF^(m/m) than in VEGF^(+/+) mice (capillaries/mm²: 1200±150 inVEGF^(+/+) mice versus 1740±150 in VEGF^(m/m) mice; n=5; p<0.05).Fluoro-angiography⁶ of the diaphragm revealed comparable vascularizationin both genotypes. (ii) Sciatic nerves in VEGF^(m/m) mice had a similardensity of vasa nervorum (capillaries/mm²: 90±6 in VEGF^(+/+) miceversus 100±7 in VEGF^(m/m) mice; n=7; p=NS), were normally perfused(laser doppler blood flow perfusion units: 150±15 in VEGF^(+/+) miceversus 190±12 in VEGF^(m/m) mice; n=5; p=NS), and contained a comparabledensity and pattern of peri- and endoneural vessels without signs ofleakiness or obstruction (fluoro-angiography). Axonal degenerationoccurred in the periphery as well as in the center of VEGF^(m/m) nerves,arguing against ischemic neuropathy, which is typically more severe inthe center³⁷. (iii) In the spinal cord, capillary densities werecomparable in both genotypes in the gray matter (capillaries/mm²: 380±17in VEGF^(+/+) mice versus 390±13 in VEGF^(m/m) mice; n=7; p=NS), and inthe white matter (capillaries/mm²: 170±12 in VEGF^(+/+) mice versus170±11 in VEGF^(+/+) mice; n=7; p=NS), with similar densities in ventraland dorsal horns. Endothelial cells of both genotypes expressedblood-brain barrier characteristics (glucose-transporter type I;Glut-1). Signs of ischemic or diabetic microangiopathy³⁸ were notdetected in VEGF^(m/m) mice. Characteristic signs of ischemic neuropathy(amyloid deposits, inflammatory vasculitis) or diabetic neuropathy(hyalinization of endoneural microvessels, thickening of capillarybasement membrane, pericyte drop-out, lumen obstruction due toendothelial hyperplasia/hypertrophy, neovascularization, nerve infarct)were not deteced in VEGF^(m/m) mice. The muscle weakness in VEGF^(m/m)mice was not due to impaired oxygenation, nor to reduced levels of theO₂-carrier hemoglobin (normal hematological profile). In addition,echocardiographic determination of the circumferential fiber shortening(VC_(F), a measure of contractility) revealed that VEGF^(m/m) mice hadnormal cardiac function. during baseline conditions (15±2 in VEGF^(+/+)mice versus 17±3 in VEGF^(m/m) mice; p=NS) and after dobutamine-stress(27±6 in VEGF^(+/+) mice versus 26±6 in VEGF^(m/m) mice; p=NS). Therewas also no metabolic imbalance in mutant mice (normal electrolytes;plasma glucose levels: 200±10 mg/dl in VEGF^(+/+) mice versus 180±16 inVEGF^(m/m) mice, n=7; p=NS).

After exposure to hypoxia (10% O₂; 24 h), motor neurons in bothgenotypes stained comparably for the hypoxia-marker pimonidazole. Themuscle weakness in VEGF^(m/m) mice was not due to impaired oxygenation,anemia, metabolic imbalance, cardiac dysfunction, or abnormal vasculardevelopment in other organs. Collectively, there were no signs ofvascular insufficiency or ischemia in VEGF^(m/m) mice.

8. Expression of VEGF and Neuropilin4 in the Spinal Cord

Exposure of VEGF^(+/+) mice to hypoxia (10% O₂; 24 h) upregulatedexpression of VEGF in the spinal cord (pg/mg protein: 15±1 in normoxiaversus 94±20 in hypoxia; n=7, p<0.05), but only minimally in VEGF^(m/m)mice (pg/mg protein: 9±2 in normoxia versus 15±2 in hypoxia; n=7;p=0.06). Hypoxic induction of LDH-A (another hypoxia-inducible gene) inVEGF^(m/m) spinal cords was not abrogated (LDH-A/10³ hprt mRNA copies:230±100 during normoxia versus 1100±400 during hypoxia; n=7; p<0.05),confirming specificity of gene targeting. Similar results were obtainedin the brain. In contrast, VEGF levels were reduced in skeletal muscleafter hypoxia (pg/mg protein: 40±10 in normoxia and 22±9 in hypoxia inVEGF^(+/+) mice, n=7, p<0.05 versus 52±6 in normoxia and 23±4 in hypoxiain VEGF^(+/+) mice, n=7; p<0.05), consistent with previous observationsthat VEGF expression in response to hypoxia is tissue-specific³⁹.

9. Neurotrophic Role of VEGF and Neuropilin-1

Apoptosis of neuronal cells has been implicated in severalneurodegenerative disorders, including ALS^(24,33). A possibleneuroprotective role of VEGF, independently of its angiogenic effect,was studied using NSC34 cells, a murine motor neuron cell line⁴⁰, inresponse to various apoptotic stimuli. Known motor neuron survivalfactors (bFGF⁴¹, TGFβ-1⁴²) protected NSC34 cells against TNF-α-inducedapoptosis (FIG. 5 a). Physiological concentrations of VEGF₁₆₅ alsoprotected motor neurons against apoptosis induced by TNF-α, hypoxia,oxidative stress (H₂O₂), and serum deprivation. Notably, VEGF₁₂₁ (whichdoes not bind NP-1¹⁰) did not rescue motor neurons. Involvement of NP-1was demonstrated by the partial neutralization of the VEGF₁₆₅ survivaleffect by antibodies, blocking NP-1 but not by antibodies blocking NP-2.The neurotrophic effect of VEGF₁₆₅ was also partially blocked byantibodies to VEGFR-2 but not to VEGFR-1, while complete neutralizationwas achieved by the combination of both VEGR-2 and NP-1 antibodies. NP-1is known to bind semaphorin III/collapsin-1 (Sema3A), implicated inrepulsion and patterning of sensory and motor projections in the spinalcord during development¹¹⁻¹⁵. Sema3A was recently suggested to promoteapoptosis of sympathetic and cerebellar neurons⁴³, and to prevent axonalregeneration after nerve injury in the adult⁴⁴ (and, therefore, could beimplicated in axon retraction and motor neuron death in VEGF^(m/m)mice), Exposure of wild type mice to hypoxia (10% O₂; 24 h) slightlyincreased expression of Sema3A in the spinal cord. SCN34 motor neuronsalso expressed Sema3A. Thus, motor neurons express both aneuroprotective factor (VEGF₁₆₅) as well as a neurorepulsive/apoptoticfactor (Sema3A), that are reciprocal antagonists for binding to NP-1.

10. Abnormal Neuronal Perfusion in VEGF^(m/m) Mice

We examined whether the muscle weakness and neuropathy were caused byvascular insufficiency. In VEGF^(m/m) skeletal muscle, only a reductionin capillary lumen size could be detected, but microvascular partialoxygen pressure measurements revealed that the smaller capillaries didnot cause muscle ischemia. Importantly, the capillary-to-muscle ratiowas normal when the first signs of neurogenic muscle atrophy developed,showing that impaired angiogenesis was not the cause of motor neurondegeneration. Instead, the slight decrease of this ratio in oldVEGF^(m/m) mice with severe muscle atrophy beyond 7 months may be theresult of muscle denervation, as observed in patients with denervationmuscle atrophy (Carpenter et al. (1982) Muscle Nerve 5, 250).Furthermore, PCNA-labeling failed to detect genotypic differences inendothelial proliferation in skeletal muscle at all ages andfluoro-angiography (Schratzberger P. et al., (2000) Nat Med 6, 405) ofthe diaphragm revealed comparable vascularization in both genotypes. Noobvious structural vascular defects could be detected in neuronal tissuebut, surprisingly, neuronal perfusion was reduced by 50% in VEGF^(m/m)mice. In sciatic nerves, both genotypes had a comparable density of vasanervorum (capillaries/mm²: 90±6 in VEGF^(+/+) mice versus 100±7 inVEGF^(m/m) mice; n=7; p=NS) and pattern of peri- and endoneural vesselswithout signs of leakiness or obstruction (fluoro-angiography). In thespinal cord, capillary densities were comparable in both genotypes inthe gray matter (capillaries/mm²: 380±17 in VEGF^(+/+) mice versus390±13 in VEGF^(m/m) mice; n=7; p=NS) and in the white matter(capillaries/mm²: 170±12 in VEGF^(+/+) mice versus 170±11 in VEGF^(m/m)mice; n=7; p=NS), with similar densities in ventral and dorsal horns.Endothelial cells in VEGF^(m/m) mice expressed blood-brain barriercharacteristics (glucose-transporter type I; Glut-1), butultrastructural signs of diabetic microangiopathy (Boulton A. J. et al.(1998) Med Clin North Am 82, 909) were not detected. Because of theinaccessibility and small size of the spinal cord, blood flow wasquantified in the brain using microspheres. Baseline cerebral blood flowwas 0.9±0.1 ml/min/g in VEGF^(+/+) mice versus 0.5±0.1 in VEGF^(m/m)mice (n=8; p<0.05). VEGF^(m/m) mice were, however, still able toincrease their cerebral blood flow in response to hypercapnia (7.5%CO₂), as measured using laser doppler (% increase of flow: 43±3% inVEGF^(+/+) mice versus 39±6% in VEGF^(m/m) mice; n=10; p=NS). Theneuronal perfusion deficit appeared to be specific as renal perfusionwas normal in VEGF^(m/m) mice (1.5±0.2 ml/min/g in VEGF^(+/+) miceversus 1.8±0.3 in VEGF^(m/m) mice; n=8; p=NS). It should be clear thatcharacteristic signs of diabetic neuropathy (hyalinization of endoneuralmicrovessels, thickening of capillary basement membrane, pericytedrop-out, lumen obstruction due to endothelial hyperplasia/hypertrophy,neovascularization, nerve infarct) were not detected in VEGF^(m/m) mice.Furthermore, the muscle weakness in VEGF^(m/m) mice was not due toimpaired oxygenation, nor to reduced levels of the O₂-carrier hemoglobin(normal hematological profile). In addition, echocardiographicdetermination of the circumferential fiber shortening (VC_(F), a measureof contractility) revealed that VEGF^(m/m) mice had normal cardiacfunction during baseline conditions (15±2 in VEGF^(+/+) mice versus 17±3in VEGF^(m/m) mice; p=NS) and after dobutamine-stress (27±6 inVEGF^(+/+) mice versus 26±6 in VEGF^(m/m) mice; p=NS). There was also nometabolic imbalance in mutant mice (normal electrolytes; plasma glucoselevels: 200±10 mg/dl in VEGF^(+/+) mice versus 180±16 in VEGF^(m/m)mice, n=7; p=NS). In conclusion, the muscle weakness and neuropathy inVEGF^(m/m) mice were not due to reduced oxygen saturation levels in theblood, anemia, metabolic imbalance or cardiac dysfunction.

Materials and Methods. 1. Generation of VEGF^(m/m) Mice

The murine VEGF gene (129/SvJ; Genome Systems Inc., St. Louis, Mo.) wasisolated and mapped previously²⁶. Deletion of the HIF-1alfa binding sitein the VEGF promoter was achieved by constructing a targeting vector,pBSK.VEGF^(m), in which the wild type TACGTGGG HIF-1alfa responseelement (HRE) was deleted, which abolishes HIF-1alfa binding²³. Thisvector contained a neomycin phosphotransferase (neo) cassette, flankedby IoxP sites to allow subsequent removal by Cre-recombinase (FIG. 1 a).After electroporation of pBSK.VEGF^(m), recombined ES cell clones,containing both the HIF-1alfa binding site deletion and the floxedneo-cassette (VEGF^(+/neo)) were identified by Southern blot analysisand sequencing (FIG. 1 a). VEGF^(neo/neo) ES cells were obtained byculturing VEGF^(+/neo) ES cells in high G418 selection (1800 μg/ml), andused to obtain VEGF^(m/m) ES cells by transient expression of the Crerecombinase. Probes for Southern blot analysis included: probe A (0.7 kbPst1/BstEII fragment) and probe B (1 kb PCR fragment, amplified fromgenomic DNA, using as forward primer 5′ TTA TCA GAA TTC ATT CCC GAG GCCTGG GGA GAG TTG GG-3′ and as reverse primer 5′-ATA AAG AAT TCG GAA GGTCAC AGC CCT TCG GTG G-3′). Analytical restriction digests used foridentification of recombinant ES cell clones are indicated. TargetedVEGF^(+/neo) ES clones were used to generate chimeric mice via morulaaggregation, that were testbred with Swiss females for germlinetransmission. Viable VEGF^(+/neo) offspring were not obtained,presumably because the presence of the neo-gene inactivated VEGF geneexpression and caused haploinsufficient lethality. However, whenchimeric mice were intercrossed with pgk:Cre mice, viable VEGF^(+/m)offspring were obtained, that were intercrossed to obtain homozygousVEGF^(m/m) offspring. All methods of ES culture, selection, and diploidaggregation have been described²⁶.

2. Gene Expression, Morphology, Motor Performance Tests, Torque andElectromyography

Western and Northern blotting, quantitative real-time RT-PCR, histology,electron microscopy, immunostaining, alone or in combination with insitu hybridization, and morphometric analysis were performed aspreviously described^(4,26,56). The following antibodies were used forimmunostaining: Glut-1 (C-20; Santa Cruz Biotechnology Inc, Santa Cruz,Calif.), VEGF (Santa Cruz), desmin (D33; Dako S/A, Glostrup, Denmark),ChAT (AB144; Chemicon, Biognost, Wevelgem), NF (SM32; SternbergerMonoclonals Inc., Lutherville, Md.), NF_(P) (SMI 31; SternbergerMonoclonals Inc.), calretinin (Swant, Bellinzon, Switzerland), MAP2(Sigma, Bornem, Belgium), GFAP (Z0334; Dako S/A), ubiquitin (Z0458; DakoS/A), synaptophysin (A0010; Dako S/A), F4/80 (A3-1; Serotec Ltd, Oxford,UK), pimonidazole hydrochloride (Hypoxyprobe-1; Natural PharmaciaInternational Inc., Belmont, Md.), BrdU (Beckton Dickinson, Brussels,Belgium). Histochemical staining (myosin ATPase, Nissl, Bielschowski)was performed using standard protocols. All stainings were performed on7 μm-thick sections, except for ChAT (40 μm), Nissl (15 μm) and myosineATPase (15 μm). Quantitative real-time RT-PCR analysis was performed aspreviously described⁵⁶. The relative expression levels of these geneswere calculated by dividing their signals by the signals obtained forthe HPRT gene.

Motor coordination and muscular performance tests (footprint test,hanging test, grip test, rotating axle test)²⁹, electromyographicrecordings in anesthetized mice⁵⁸, and echocardiographic analysis⁵⁶ wereperformed as described. All animal procedures were approved by theethical committee. For fluoro-angiography⁶, 500 μl of 5% fluorescentdextran (molecular weight of 2×10⁶ dalton; Sigma) was injectedintravenously in urethane-anesthetized mice. After 5 minutes, mice wereperfused with 1.9 ml fluorescent dextran and 100 μl adenocor (SanofiPharma, Brussels, Belgium), and sciatic nerves were immediately analyzedby confocal microscopy. Laser-doppler measurements of blood flow (bloodperfusion units) through sciatic nerves was performed in anesthetizedmice using a needle flow probe (ADInstruments Pty Ltd, Castle Hill,Australia) at 1 mm intervals across a 5 mm nerve segment. Analysis ofblood gases, clinical chemistry and hematologic profile was performedusing standard techniques at the University Hospital (Leuven, Belgium).

3. Cell Culture and Survival Analysis

SCN-34 cells were cultured as described⁴⁰. Blocking antibodies to NP-1and NP-2 were a gift from Dr. A. Kolodkin, and VEGFR-2 antibodies(DC101) from Dr. P. Bohlen, (Imclone). For apoptosis studies, SCN-34cells were cultured in 175 flasks coated with 0.1% gelatin in RPMI 1640medium containing 10% foetal calf serum (Life Technologies, Paisley,UK), 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine,heparin (100 μg/ml) and endothelial cell growth supplement (30 μg/ml).Apoptosis was induced by supplementation of TNF-alfa (50 ng/ml; R&D,Abingdon, UK), withdrawal of growth factors (0.1% or 0.5% fetal calfserum), or treatment with hypoxia (2% O₂) or 2% H₂O₂. VEGF₁₂₁ andVEGF₁₆₅ were from R&D. Apoptosis was quantified by measuring cytoplasmichistone-associated DNA fragments (mono- and oligonucleosomes) using aphotometric enzyme immunoassay (Cell Detection ELISA, BoehringerMannheim, Mannheim, Germany). Determination of VEGF levels was performedusing commercially available ELISAs (R&D).

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1-15. (canceled)
 16. A method of treating a neuron disorder in a mammal,the method comprising administering to the mammal a VEGF protein. 17.The method of claim 16, wherein the neuron disorder is a neuropathy. 18.The method of claim 17, wherein the neuropathy is a motor neurondisorder.
 19. The method of claim 18, wherein the motor neuron disorderis amyotrophic lateral sclerosis (ALS) or an ALS-like disorder.
 20. Themethod of claim 16, wherein the VEGF is selected from the groupconsisting of VEGF-A and VEGF-B.
 21. The method of claim 20, wherein theVEGF-A is isoform VEGF165.
 22. The method of claim 16, wherein the VEGFis adminstered intrathecally.
 23. A method of enhancing survival of amotor neuron in a mammal, the method comprising contacting a motorneuron of the mammal with an amount of a VEGF protein effective toenhance survival of the motor neuron.
 24. The method of claim 23,wherein the motor neuron is hypoxic.
 25. The method of claim 23, whereinthe motor neuron is contacted in vitro.
 26. The method of claim 23,wherein the VEGF is selected from the group consisting of VEGF-A andVEGF-B.
 27. The method of claim 26, wherein the VEGF-A is isoform VEGF165.
 28. The method of claim 23, wherein contacting the motor neuron isthrough intrathecal administration.
 29. A method of treating a neurondisorder in a mammal, the method comprising administering to the mammala nucleic acid sequence encoding VEGF.
 30. The method of claim 29,wherein the VEGF is selected from the group consisting of VEGF-A andVEGF-B.
 31. The method of claim 30, wherein the VEGF-A is isoform VEGF165.
 32. A method of enhancing survival of a motor neuron in a mammal,the method comprising contacting a motor neuron of the mammal with anucleic acid sequence encoding an amount of a VEGF protein effective toenhance survival of the motor neuron.
 33. The method of claim 32,wherein the motor neuron is hypoxic.
 34. The method of claim 32, whereinthe motor neuron is contacted in vitro.
 35. The method of claim 32,wherein the VEGF is selected from the group consisting of VEGF-A andVEGF-B.
 36. The method of claim 35, wherein the VEGF-A is isoform VEGF165.
 37. A method for identifying a molecule which stimulates survivalof motor neurons, wherein the method comprises exposing a neuropilin-1receptor and a VEGF Receptor-2 to at least one molecule thought tomodulate motor neurons, and monitoring survival of the motor neurons.38. The method of claim 37, wherein said monitoring comprisingdetermining inhibition of semaphorin 3A-induced death of the motorneurons.
 39. A method of producing a pharmaceutical compositioncomprising mixing a molecule identified as stimulating survival of motorneurons with a pharmaceutically acceptable carrier.